It is well known in the art of discovering novel chemical compounds with therapeutic effects that plants have yielded some of the most important molecules in history. Civilizations around the globe have exploited the medicinal benefits of plants for millennia. Today, private, public, and government institutions devote extensive resources searching for molecules in plants that may have a potential economic and humanitarian impact. Technological advances in laboratory automation, biochemistry, and molecular biology enable us to currently screen hundreds of thousands of molecules for biological activity every day.
Cragg, et al., in “The search for new pharmaceutical crops: Drug discovery and development at the National Cancer Institute,” 161-167, describe the extensive natural library testing program of the National Cancer Institute (NCI) and the methods used to prepare plant extracts for screening. Cardellina II, et al., in J. Nat. Prod., 56(7), 1123-1129 (1993), describe the screening program of the NCI and also specifically discuss the chemical interferences ubiquitous within plants and current techniques used to remove these chemicals before screening or after screening. Turner, in J. Ethnopharm., 51, 39-44 (1996), describes screening plants at a large pharmaceutical company. Borris, in J. Ethnopharm., 51, 29-38 (1996), describes the increased complexities that come with screening plant extracts using a competitive screening program that requires a structured approach and the latest scientific techniques. Shu, in J. Nat. Prod., 61, 1053-1071 (1998), promotes the value of novel chemicals that have been isolated from plants, and lists points a screening laboratory must achieve to improve the success rate when testing plant extracts. The points on the list are not easily accomplished and include challenges such as making a screen suitable for natural libraries, removing all interferences, and accelerating dereplication.
Preparing plant extracts for screening has always been recognized as laborious, and published literature suggests that the method of preparation is more important than previously and currently understood. Plants have numerous ubiquitous compounds that may mask an effect or interfere with the mechanism of action of a biological assay. Ingkaninan, J. Nat. Prod., 62(6), 912-914 (1999), Kato, J. Steroid Biochem., 34(1), 219-227 (1989), Vallete et al., in Endocrin., 129(3), 1363-1369 (1991), and Kang et al, in Biochem J., 303, 795-802 (1994), have reported that fatty acids, phospholipids, and tri-, di-, and monoglycerides cause noncompetitive or mixed noncompetitive inhibition on some receptors or modify the structure or confirmation of receptors in cell-based assays. Numerous plant solvent extracts have high molecular weight compounds that make up greater than 70% of the mass of the extract and that never could be approved drugs. Numerous laboratories do not remove or have not removed these compounds before screening. This may result in false positives or false negatives during subsequent biological assays. Tan, et al., in J. Nat. Prod., 54(1), 143-154 (1991), Cardellina II, et al., in J. Nat. Prod., 56(7), 1123-1129 (1993), Claeson, et al., in J. Nat. Prod., 61(1), 77-81 (1998), Lee, et al., in J. Nat. Prod., 61(11), 1407-1409 (1998), and Patil, et al., in J. Nat. Prod., 60(3), 306-308 (1997), describe false positives that may be attributed to polyphenols and tannins. Some laboratories remove these compounds before screening, while others remove these compounds only after a potential false positive has occurred, believing that these compounds cannot cause a false negative. Phillipson, in J. Pharm. Pharmacol. 51:493-503 (1999), has suggested further that partially purified plant extracts without common metabolites may prove attractive to screening laboratories.
A general mantra in preparing plant extracts states, “it is not what you miss, but what you hit.” This approach has led laboratories to put ease of preparation and number of plant extracts prepared and screened ahead of a scientifically based approach for success. Laboratories typically prepare one to three extracts per plant for screening. These extracts may contain one hundred to thousands of chemical compounds per extract.
Because the collision frequency and proper orientation of a ligand and its receptor play a role in binding, screening plant extracts with numerous compounds may interfere with the detection of potential biological effects. In addition, increased dipole-dipole interactions, hydrogen bonding, or steric effects that exist in physiological conditions could also contribute to the disruption of ligand binding. Haberlein, in Planta Medica, 62(3):227-31 (1996), indicate that two different concentrations of the same ethanolic plant extract cause positive and negative allosteric regulation of a GABA receptor. In contrast to accepted principles, interferences may result in false positives as well as false negatives. Menzies, in Eur. J. Pharm., 350(1), 101-108 (1998), suggests that the bioactivity of a known compound in a plant extract is not observed in an opioid assay because of an interfering compound canceling out its activity. Phillipson, in J. Pharm. Pharmacol. 51:493503 (1999), states that the activity of an isolated compound is not always directly comparable to the plant extract from which it was isolated. These suggestions, empirical data, and hypotheses show that many variables exist in the screening process. Because the process of scientific investigation and discovery should reduce the number of variables and operate in a closed system, the removal of all potential interferences from a plant extract before entering a biological screen and further separation of the drug-like chemical compounds to further reduce interference and enable each chemical compound to be tested at its detectable screening concentration are essential.